How is the result of a direct ELISA measured?

The procedure for carrying out a direct ELISA involves an antigen or substance being immobilized directly on the microplate. A primary antibody is then added to bind to the target protein. After washing the plate, a substrate/chromophore (e.g. AP or HRP) is subsequently added, generating a signal (color change) that is directly correlated with the quantity of analyte in the sample. The color change happens due to the oxidation of substrates by horseradish peroxidase or via hydrolysis of phosphate groups of substrates by alkaline phosphatase. A microplate reader is then used to quantify the absorbance of the results, typically at 450 nm with a reference wavelength at 655 nm. The data in the microplate reader is displayed in a graph of optical density vs the log concentration of the sample. To measure the concentration of an analyte, a standard curve is required produced from a dilution of a known concentration of antigen. The absorbance of each signal per sample is compared to the standard curve to calculate the concentration of the analyte. When doing this, the mean absorbance should be taken from the samples and plotted on the Y axis against known protein concentration of the standards on the X axis. A best fit curve should then be drawn to connect the data points on the graph. Next, a horizontal line should be drawn from the Y axis to the standard curve. At the point of contact between the absorbance and standard curve, a vertical line should be drawn straight down until it reaches the X axis to determine the correlating concentration of the analyte. It is important to note that the concentration must be multiplied by the dilution factor of the sample for accurate results.